Monitoring Chemical and Biological Electron Transfer Reactions with

Subscriber access provided by Fudan University

Article

Monitoring Chemical and Biological Electron Transfer Reactions with a Fluorogenic Vitamin K Analogue Probe Mei Ni Belzile, Robert Godin, Andres M. Durantini, and Gonzalo Cosa J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b09735 • Publication Date (Web): 22 Nov 2016 Downloaded from http://pubs.acs.org on November 22, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of the American Chemical Society is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 12

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of the American Chemical Society

Mei-Ni Belzile, Robert Godin, Andrés M. Durantini, and Gonzalo Cosa* Department of Chemistry and Center for Self-Assembled Chemical Structures, McGill University, 801 Sherbrooke Street West, Montréal, Québec, H3A 0B8, Canada. ABSTRACT: We report herein the design, synthesis and characterization of a two-segment fluorogenic analogue of vitamin K, B-VKQ, prepared by coupling vitamin K3 also known as menadione (a quinone redox center) to a boron-dipyrromethene (BODIPY) fluorophore (a lipophilic reporter segment). Oxidation-reduction reactions, spectroelectrochemical studies and enzymatic assays conducted in the presence of DT-diaphorase illustrate that the new probe shows reversible redox behavior on par with that of vitamin K, provides a high sensitivity fluorescence signal, and is compatible with biological conditions, opening the door to monitor remotely (i.e. via imaging) redox processes in real-time. In its oxidized form, B-VKQ is nonemissive, while upon reduction to the hydroquinone form, B-VKQH2 , BODIPY fluorescence is restored with emission quantum yield values of ca. 0.54 in toluene. DFT studies validate a photoinduced electron transfer (PeT) intramolecular switching mechanism, active in the non-emissive quinone form and deactivated upon reduction to the emissive dihydroquinone form. Our results highlight the potential of B-VKQ as a fluorogenic probe to study electron transfer and transport in model systems and biological structures with optimal sensitivity and desirable chemical specificity. The use of the probe may enable a better understanding of the role that vitamin K plays in biological redox reactions ubiquitous in key cellular processes, and help elucidate the mechanism and pathological significance of these reactions in biological systems.

INTRODUCTION

vitamin K redox and transport properties are highly desirable. A fluorogenic probe15-21 capable of reporting via emission enhancement changes in the redox status of vitamin K is essential towards gaining a mechanistic understanding of vitamin K’s physiological role. This probe when coupled with state-of-the-art microscopy imaging technologies, will enable interrogating the redox status of biological systems with high spatiotemporal resolution. Such a tool will offer the opportunity to correlate electron transport activity with cell homeostasis, and to develop diagnostics of mitochondrion activity and mitochondrial diseases associated to deficiencies in electron transport. Ultimately, a fluorogenic vitamin K analogue may shed light on the overall impact of this vital carrier in sustaining cell viability.

Electron transport within cell membranes is a most fundamental chemical process in living systems, involved in cellular respiration and in photosynthesis. Deficiencies in electron transport during respiration are associated with a number of pathologies and to the onset of oxidative stress and ROS generation.1,2 Electron transport requires interplay between a number of protein complexes and their associated redox cofactors, and for membrane-embedded mobile carriers acting as electron relay systems to transport electrons between complexes along the lipid membrane.3-5 Carriers may involve a quinone/dihydroquinone redox couple operating via proton-coupled electron transfer (PCET) reactions.6

Building on our own experience in preparing lipophilic fluorogenic probes including α-tocopherol21-24 analogues and a recent ubiquinone25 analogue, here we report the design, synthesis and characterization of a fluorogenic probe, B-VKQ (Figure 1C), which mimics vitamin K in structure and activity. B-VKQ is a two-segment receptor-reporter probe26 consisting of the redox-sensitive menadione moiety (2-methyl-1,4-naphthoquinone, vitamin K3) coupled to the meso position of a BODIPY fluorophore via a methylene linker. The probe relies on intramolecular photoinduced electron transfer from the naphthoquinone segment (reporter) to the BODIPY moiety (receptor) to impart an off-on fluorescence switch. In the oxidized form, B-VKQ is

Ubiquitously found in Nature, compounds within the vitamin K family7 which bear a 2-methyl-1,4-naphthoquinone core and a variety of alkyl tails (Figure 1) act as electron carriers in bacterial cellular respiration8,9 and in the photosynthetic membrane.10,11 Vitamin K2 has further been shown to serve as a mitochondrial electron carrier in Drosophila.9 Additionally vitamin K is an important cofactor in blood coagulation of mammals8,12 where it plays a pivotal role as a redox relay molecule in the gamma-carboxylation of specific glutamate residues in vitamin K-dependent proteins.13,14 Given its critical role in living systems, non-invasive imaging methodologies able to monitor real-time and in situ 1

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 12

exploited to switch the probe emission.21,26,29 By tuning the BODIPY segment only while preserving intact the receptor (vitamin K) segment, we ensured retaining the intrinsic chemical properties of the latter.23,24 Together, the redox active menadione segment and the judiciously chosen lipophilic fluorescent segment rendered a fluorogenic analogue of vitamin K.

completely non-emissive. Upon reduction of the menadione segment to menadiol, the highly emissive dihydroquinone form of the probe B-VKQH2 is generated with an emission quantum yield Φf of ca. 0.54. The menadione-BODIPY adduct emulates the redox properties and chemical reactivities of vitamin K species, preserving their lipophilic character and preferential lipid partitioning, while yielding a highly sensitive, reversible redox probe19 reporting a two electron and two proton reduction via dramatic emission enhancement both in non-polar but also polar solvents such as acetonitrile. In this regard B-VKQ amply surpasses the sensitivity performance of a recently published fluorogenic analogue of ubiquinone,25 characterized by a slightly lower redox potential.

To ensure that PeT from the BODIPY to the naphthoquinone segment would be exergonic30 (non-emissive) in the oxidized form of the naphthoquinone/menadione segment but endergonic (emissive) in its reduced form, we chose electron releasing alkyl groups to decorate the BODIPY core. These groups raise both the HOMO and LUMO of the BODIPY segment enhancing the feasibility of PeT from the BODIPY to naphthoquinone while preventing PeT from the HOMO of the reduced form of naphthoquinone to the BODIPY core.30 Density functional theory (DFT) calculations at the B3LYP 6-31g(d) level theory31 confirmed that alkyl group substitution on the BODIPY core satisfied these criteria, rendering a dark compound in its oxidized form and an emissive one following reduction, see Figure 2. Additionally, alkyl substitution in BODIPY dyes increases their lipophilic character.32

Oxidation-reduction reactions, spectroelectrochemical studies with trace amounts of compound and enzymatic assays conducted in the presence of DT-diaphorase illustrate that the new probe shows reversible redox behavior on par with that of vitamin K, provides a high sensitivity fluorescence signal, and is compatible with biological conditions. The new probe opens the door to monitor remotely (i.e. via imaging) redox processes in real-time, and potentially at the single molecule level.

In order to retain high lipophilicity, maximize structural similarity to menadione, and minimize the distance between the vitamin K and BODIPY moieties, while ensuring competitive PeT in the oxidized form, a short aliphatic methylene bridge was incorporated in the design.23 Synthesis of B-VKQ. We used the commercially available 1,4-dihydroxy-2-naphthoic acid (1) as the precursor of the vitamin K/menadione receptor segment. To introduce the methylene linker between the menadione and BODIPY segments, the aliphatic chain of the starting carboxylic acid 1 had to be extended by one carbon (Scheme 1). After protection of the sensitive hydroxyl groups, the methyl ester functionality was reduced to an alcohol, and then replaced with a bromine atom. Substitution by a nitrile and hydrolysis under basic conditions yielded the desired carboxylic acid, 1,4-dimethoxy-2-methylnaphthoic acid (2 in Scheme 1). The acid chloride of 2 was then prepared, and reacted with 2 equivalents of 3-ethyl-2,4-dimethylpyrrole in the presence of phosphorus oxychloride. Pyrrole condensation gave the dipyrrin bearing the desired vitamin K substituent at the meso position. Subsequent deprotection with BBr3, followed by addition of DIPEA and BF3·OEt2 gave the desired B-VKQ probe in 8% overall yield starting from compound 2 (since the deprotected hydroquinone rapidly oxidizes in air, we obtained the probe, B-VKQ in the quinone form.). The methyl-protected form of the probe, B-VKQMe2 was also obtained by simply omitting the deprotection step with BBr3 (Scheme 1).

Figure 1. Structures of A) natural form of lipophilic vitamin K1 B) menadione (also known as vitamin K3) and C) the fluorogenic vitamin K analogue, B-VKQ reported herein.

RESULTS AND DISCUSSION Design of B-VKQ. In order to provide chemical specificity and redox reversibility characteristic of vitamin K, we selected the menadione moiety as the receptor segment. A BODIPY chromophore was used as the reporter segment. BODIPYs have optimal spectroscopic properties and their lipophilic nature enables substitution of the alkyl tails of vitamin K1 and K2 for the dye without altering the lipid partitioning (Figure 1). BODIPY fluorophores are also amenable to synthetic modifications to tune their redox properties.27,28 Photoinduced electron transfer (PeT) may then be

2


Page 3 of 12

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. Off/on fluorogenic mechanism of B-VKQ based on PeT. HOMO (black line) and LUMO (red line) energy levels for receptor (vitamin K) and reporter (BODIPY) moiety of the oxidized and reduced form of the B-VKQ probe as calculated computationally using B3LYP 6-31g(d) level theory without solvent model. Scheme 1. Preparation of B-VKQ.

Spectroscopic and Electrochemical properties of B-VKQ. The absorption and fluorescence spectroscopic properties of B-VKQ, B-VKQH2 , and B-VKQMe2 are presented in Table 1 (see Figure s1 for structures and Figure S2 for spectra). While the quinone form is non-emissive regardless of the solvent, both the hydroquinone and methoxy protected forms are emissive, where their quantum yields are 5 to 10-fold smaller in acetonitrile than toluene. In turn time-resolved fluorescence experiments revealed that the decay rate constant kdec of the excited sate is 3-7 fold faster in the more polar solvent, primarily caused by a larger non-radiative rate constant (see Table 1). The increase in non-radiative decay may be attributed to an in-

tramolecular quenching pathway, presumably photo-induced reduction of the BODIPY chromophore by the dihydroquinone moiety or its methyl ether form, as seen by the proximity in energy of the BODIPY and dihydroquinone HOMOs (Figure 2). This process is facilitated in polar (e.g. acetonitrile) solvents.33,34 In order to show the electrochemical reversibility of the B-VKQ/B-VKQH2 redox couple cyclic voltammetry (CV) was next performed in dry acetonitrile and subsequently in the presence of 10.8 M water, see Table S1 for a list of the redox potentials recorded.

3



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 12

Table 1. Spectroscopic properties of B-VKQMe2 , B-VKQ, and B-VKQH2 in various solvents. ε ×103

kdec × 107

krad × 107

knr × 107

(s-1)

(s-1)

(s-1)

0.16±0.01

68.9

11.0

57.9

80

0.87±0.02

18.4

16.2

2.2

21c

0.00070±0.00002

–b

–b

–b

75

0.0006±0.0002

–b

–b

–b

536

35±1d,e

0.0400±0.0001d,e

142.8d,e

5.7

137.1

541

85±5e

0.54±0.05e

21.1e

11.4

9.7

Compound

Solvent

Abs λmax (nm)

Em λmax (nm)

(M-1 cm-1)

B-VKQMe2

MeCN

523

538

38c

Toluene

528

540

MeCN

525

544

Toluene

532

546

B-VKQ B-VKQH2

MeCN Toluene

522 529

Φf a

aQuantum

yields were measured using PM605 as a standard (ϕf = 0.72 and 0.89 in acetonitrile and toluene, respectively).36 bNot available. cAlthough we do not fully understand the cause of the substantial decrease in extinction coefficient in acetonitrile, it is possible that aggregation and subsequent distortions to the electronic structure are at play dSpectroscopic measurements of B-VKQH2 in acetonitrile were performed by carrying out in situ reduction of B-VKQ with sodium borohydride in toluene and then diluting a 10 μL aliquot of this reaction mixture in 3 mL of acetonitrile. The direct reduction in acetonitrile led to undesired BODIPY decomposition (Figure S7). eWe assume the in situ reduction achieves almost complete conversion to the hydroquinone form B-VKQH2 and thus the amount of starting B-VKQ is negligible.

In aprotic conditions (specifically in dry acetonitrile which likely contains traces of water), we observed two cathodic peaks in the CV experiment for B-VKQ; a first reversible peak at –0.40 V and a second quasi reversible peak at –1.00 V vs. NHE (Figure 3A, showing 6 scans). The first peak at –0.40 V was assigned to the first reduction of the naphthoquinone segment to its semiquinone radical anion form, consistent with literature values for the first standard 𝑜 ∙− = –0.41 reduction potential for menadione given as 𝐸𝑄/𝑄 35 V vs NHE (in DMF and acetonitrile) and also consistent with a value of –0.53 V we recorded in acetonitrile (vide infra).

the electrochemistry of vitamin K in acetonitrile, and quinones in general, that addition of water or acid results in the coalescence of the first and second 1-electron reduction peaks recorded under anhydrous aprotic conditions into a single 2-electron reduction peak shifted to less negative bias.37-39

The second peak was assigned to a complex reduction of the coupled BODIPY core and quinone moiety. The redox potential recorded herein is similar to the reported 𝑜 ∙− = value for an analogous ethyl-substituted BODIPY (𝐸𝐵/𝐵 30,36 –1.21 V vs. NHE in acetonitrile). The cathodic peak at – 1.00V may also contain a contribution from the reduction of the semiquinone radical anion to the deprotonated dihydronaphthoquinone B-VKQ2- consistent with literature values reporting 𝐸𝑄𝑜∙− /𝑄2− = –1.14 V vs. NHE for menadione in DMF and acetonitrile35 and consistent with our own value of –1.16 V recorded in acetonitrile (vide infra). We also observed the reversible oxidation of the BODIPY moiety at 1.29 V, further consistent with previous reports (𝐸𝐵𝑜∙+/𝐵 = 1.23 V vs. NHE in acetonitrile).30,36 The redox potentials of the BODIPY oxidation and reduction of the naphthoquinone, combined with the E00 value obtained from steadystate optical spectroscopy, allow us to calculate a driving force of photoinduced electron transfer of –0.78 (see SI). Comparing this to the calculated value of –0.82 eV confirms our expectations and supports the initial assessment of probe design by computational methods. To better establish the origin of the peaks and their reversible nature, we next conducted CV experiments under protic conditions; (see Figure 3B). It has been shown for

Figure 3. A) Cyclic voltammogram (6 scans) for B-VKQ (1 mM) in argon-purged dry acetonitrile solution (likely with traces of water) containing 0.1 M Bu4NPF6. The Fc+/Fc (1mM) redox 4


Page 5 of 12

couple was used as an internal standard. B) CV for B-VKQ (6 scans) recorded with 10.8 M water, under otherwise identical experimental conditions. C) CV for menadione (6 scans) recorded under trace water conditions (black) or 10.8 M water (red). All other conditions were as above.

μM in toluene) was reduced at room temperature with sodium borohydride (1 mg/mL) and a small amount of water (1 μL/mL), acting as a hydrogen bonding source. An increasing fluorescence intensity with time was observed and was periodically monitored under photoexcitation at 503 nm. The fluorescence intensity plateaued after 10 minutes of reaction, displaying a greater than 1000-fold fluorescence enhancement once the insoluble sodium borohydride was filtered out (Figure 4). The emission spectra obtained under both reduction conditions showed a good overlap (Supplementary Figure S8). Absorption spectra before and after reaction with NaBH4 further show a slight shift in λmax (Supplementary Figure S6). In order to show the reversibility of the probe, the cuvette solution was next oxidized by adding 300 μM FeCl342 in order to reconvert B-VKQH2 to initial non-emissive B-VKQ. The fluorescence decreased to that of pristine B-VKQ confirming oxidation and redox reversibility. Finally, the NaBH4 reduction was repeated a final time in order to show that the probe can return to its emissive state after oxidation. The fluorescence once again increased after a few minutes, reaching a third of the maximum fluorescence intensity achieved in the first reduction, plausibly filtrations after every step reduced the amount of probe in solution leading to this drop in intensity, or the intensity reduction indicates chemical degradation caused by the strong redox agents used.

Experiments conducted with B-VKQ in acetonitrile supplemented with 10.8 M water showed two reversible peaks at –0.26 V and –1.08 V, respectively. The data is consistent with a shift toward more positive values for the reduction potential of the naphthoquinone moiety. It is also consistent with a decoupling of the reduction potential of the BODIPY core as exemplified both by the reversible nature of the second peak and its shift toward more negative values (rather than a shift to positive values as would have been expected for the second reduction potential of the naphthoquinone moiety after addition of water). Furthermore, a reversible BODIPY core reduction at –0.97 V was observed with B-VKQMe2 (Figure S3), supporting the quasireversible nature of the –1.00 V redox process seen for BVKQ under aprotic conditions to be caused by coupling of the BODIPY and quinone moieties. While we anticipated the first reduction wave in Figure 3B to integrate for a 2-electron process under protic conditions, the CV intensity prevented clear integrations from being made. Here both reduction waves showed similar peak intensities. Control experiments performed with menadione showed that two reversible peaks originally observed in dry acetonitrile (with trace H2O) at –0.53 V and – 1.16 V coalesced into 1 peak at –0.35 V in the presence of 10.8 M water, see Figure 3C. The new wave for menadione is significantly broadened, integrating for twice as much as either of the two reversible waves recorded under trace H2O conditions. Arguably a similar peak broadening was also experienced with B-VKQ under protic conditions.

Fluorescence (counts/s)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


We may conclude that electrochemical processes of BVKQ are reversible in protic media. Importantly, the similar redox potentials and shifts of the vitamin K CV waves in BVKQ and menadione after the addition of water indicate that the desired redox behavior of menadione is conserved in the probe.

reduction

1.2x10

6

8.0x10

5

B-VKQ B-VKQ + NaBH4

4.0x105

B-VKQ + NaBH4 + FeCl3 B-VKQ + NaBH4 + FeCl3 + NaBH4 re-reduction oxidation and re-oxidation

0.0 500

600 700 Wavelength (nm)

800

Figure 4. Emission spectrum of B-VKQ (1.2 μM in toluene, argon equilibrated) before (black, □),after reduction by sodium borohydride (orange, □), subsequent re-oxidation using 300 μM FeCl3 (black, ■) and final reduction using NaBH4 (orange, ■).

Chemical Reduction. In order to validate the fluorogenic nature of B-VKQ we first carried out a chemical reduction of the menadione segment with H2 using palladized charcoal and cyclohexene with methanol as a source of protons. After 18 h, the reaction yielded a fluorescent product, determined to be the reduced form of the probe by both 1H NMR and high resolution mass spectrometry (Figures S4 and S5 in Supporting Information). Since hydroquinones oxidize rapidly in air,40 the characterization methods showed the presence of both the reduced and oxidized form of the probe, precluding the exact determination of the reaction yield.

Fluorescence Spectroelectrochemistry. In order to correlate applied redox potential with emission intensity for B-VKQ upon its electrochemical reduction, we turned to fluorescence microscopy based spectroelectrochemistry (SEC), a powerful tool that allows for monitoring trace amounts of fluorescent molecules under electrochemical control.43-47 SEC experiments were performed on a confocal microscope setup to enable visualization of newly generated fluorescent compounds (Figure 5A). An electrochemical cell was custom-built by gluing a glass tube on top of a coverslip coated with optically-transparent platinized ITO

We next attempted an in situ chemical reduction of the probe in a cuvette using sodium borohydride,41 to minimize oxidation prior to analysis. A solution of B-VKQ (1.2 5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(working electrode). In our studies, the fluorescent products were generated under the direct reduction of 500 nM B-VKQ (in dry acetonitrile, may contain trace H2O) in the

Page 6 of 12

presence of acetic acid as a proton source, in order to produce the fluorescent hydroquinone form B-VKQH2 (Figure 5B).

Figure 5. A) SEC setup depicting B-VKQ molecules that are oxidized at the surface of the Pt/ITO WE and then diffuse through the confocal observation volume (dotted oval) and emit fluorescence. The distance between the electrode surface and the observation volume was controlled by a piezo nanopositioner. B) Reduction of B-VKQ to yield fluorescent B-VKQH2 . C) Applied electrochemical potential. The potential scan rate was 20 mV/s. D) Fluorescence trajectories of pristine B-VKQ with and without the presence of AcOH, and of B-VKQMe2 (with AcOH) in acetronitrile under influence of the biases shown in C). Solutions are argon equilibrated. Times where Eapp is more negative than – 0.35 V are highlighted in gray. The concentration of B-VKQ was 500 nM. Trajectories were acquired with the observation volume 20 µm above the electrode surface and counts calculated using 100 ms time bins. E) Change in fluorescence intensity plotted versus the applied potential. Arrows indicate the direction of the potential scan, where the increase in fluorescence occurs as the potential is scanned towards negative potential, and the decrease occurs as the potential is scanned back towards 0V.

The intensity-time trajectory vs applied bias recorded for B-VKQ in the presence of acetic acid displayed a rapid rise in fluorescence intensity with potentials more negative than –0.35 V vs NHE (red trace in Figure 5D) upon scanning the potential over time (20 mV/s) towards negative bias (Figure 5C). We assign the intensity rise to the formation of B-VKQH2 . A maximum fluorescence intensity was reached at a potential of –0.57 V, going from an initial average 2.4 counts/ms at resting potential, to an average 124 counts/ms at the maximum, yielding a ca. 70-fold fluorescence intensity enhancement.48 The maximum intensity

achieved with B-VKQ with acetic acid is comparable to that recorded with 500 nM of the control compound B-VKQMe2 under identical conditions (average 155.0 counts/ms). The latter displayed a bias-insensitive intensity-time trajectory (blue trace in Figure 5D). A control experiment with B-VKQ and no acetic acid showed that the intensity remained at background levels versus the applied potential, consistent with the non-emissive character of the B-VKQ2- dianion (deprotonated form of B-VKQH2 ) formed under these conditions. Figure S10 compares the initial (at resting potential) and final (at –0.57V vs. NHE) fluorescence intensities 6


Page 7 of 12

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


and overall intensity enhancements from all SEC experiments underscoring that a significant enhancement is only observed in the electroreduction of B-VKQ under protic conditions.

electron relay system. Here B-VKQ effectively shuttles electrons from NADPH to cytochrome c, see Scheme 2. Control studies with vitamin K3 (menadione) or with no quinones were also conducted to compare the rates of reaction.

The maximum enhancement observed was recorded at a significantly less negative reduction potential (–0.57 V) than that observed in the CV experiments for the two electron reduction of B-VKQ in aprotic conditions (peak at – 1.00 V). The potential recorded is consistent with the shift toward higher (less negative) value for the second reduction peak of menadione (–0.37 V) and B-VKQ (–0.26 V) recorded in the presence of water (Figure 3).

The reduction of cytochrome c was observed to readily take place with both menadione and B-VKQ, while reduction was relatively very slow when no naphthoquinone substrate was added. The rate constant k and activity values are listed in Table 2 (see Figure S11 and the Supporting Information section for general procedures). All other controls showed no reaction, where we tested every combination of the four components involved in the enzymatic assay: DT-diaphorase, B-VKQ (or menadione), cytochrome c, and NADPH (Figure S12 and S13). The results are consistent with B-VKQ providing a suitable substrate that readily mimics vitamin K3.

Sweeping the bias beyond the intensity maximum to more negative bias lead to an intensity decrease. This potential is considerably less negative compared to reduction potentials for similar BODIPY analogues and the BODIPY reduction potential observed for B-VKQ in the presence of water (Figure 3B).22,30 Further no decrease in fluorescence was observed for the B-VKQMe2 control compound (blue trace in Figure 5D, see Figure S3 for CV). It is possible that hydrogen bonding occurs between B-VKQH2 and AcOH in the mixture turning on a new PeT quenching pathway, or that B-VKQH2 in the presence of AcOH may be easier to reduce than either component through coupling of electronic structure. As the applied potential was scanned back towards 0V, the increase in fluorescence intensity from – 0.5 V to – 0.35 V is consistent with continued reduction of the menadione moiety at these potentials. The increase is also consistent with the end of the fluorescence-quenching reduction that took place at more negative potentials. Note that the oxidation potential of the fluorescent species is expected to shift towards more positive potentials because of protonation of the anionic species originally formed. This results in the slower decay of the fluorescence as compared to the rise (Figure 5E), since the fluorescence decrease is attributed to the diffusion-limited dispersion of fluorescent B-VKQH2 rather than electrochemical re-oxidation to the non-emissive form.

Table 2. Activity of DT-diaphorase with either vitamin K3 (menadione) or vitamin K BODIPY probe (B-VKQ). Substrate

Enzyme activity

Rate constant, k

min-1

(s-1) b

(nmol

mg-1)a,b

menadione

12.35 ± 1.4

0.05 ± 0.01

B-VKQ

7.66 ± 0.3

0.007 ± 0.001

none

3.23 ± 0.6

0.00008 ± 0.0001

anmol of cytochrome

c reduced per minute per mg of enzyme. are averages of three replicate experiments with the according standard deviation. bValues

Fluorescence monitoring of the DT-diaphorase enzyme-assisted reduction of B-VKQ to the dihydroquinone B-VKQH2 allowed a sensitive visualization of the reaction. The enzymatic assays were carried out under the same conditions as above, without the presence of cytochrome c to prevent regeneration of the oxidized form of the probe. In order to minimize BODIPY aggregation arising from π-π stacking and hydrophobic interactions in aqueous solution, potentially leading to self-quenching,55 we conducted the assay in liposomes, where the lipophilic B-VKQ embedded in the bilayer.

Altogether the previous results show that the system BVKQ / B-VKQH2 provides a reversible and most sensitive fluorescence marker of redox status. Here enhancements necessitated the formation of B-VKQH2 .

In the absence of oxygen, a larger than 30-fold fluorescence intensity enhancement was observed upon the enzymatic reduction of B-VKQ. Control experiments showed no increase in fluorescence in the absence of either B-VKQ, or NADPH or DT diaphorase enzyme (Figure S14). Recording the emission spectrum of the enzymatic reduction over time, the appearance of an emission peak at 537 nm was observed consistent with the formation of the reduced B-VKQH2 species (Figure S15). These results further support that B-VKQ probe is compatible with biological systems and may serve as an electron shuttle working in tandem with proteins.

Enzymatic Assays. To determine whether the probe is recognized as an enzyme substrate, and may act as an electron shuttle in biological systems, we performed enzymatic assays using B-VKQ and the enzyme DT-diaphorase, also known as NAD(P)H:quinone oxidoreductase (EC 1.6.99.2).49 This flavoprotein catalyzes the two electron reduction of quinones and quinoid compounds (including K vitamins) using either NADPH or NADH as electron donors.50,51 DT-diaphorase activity was first determined spectrophotometrically by measuring the increase in absorption at 550 nm upon reduction of cytochrome c in the presence of NADPH as an electron source and B-VKQ as an

7



Scheme 2. Electron shuttling by B-VKQ / B-VKQH2 where DT-diaphorase catalyzes the reduction of cytochrome c using NADPH as the electron donor and using quinones as an electron relay system. DT-diaphorase and cytochrome c structures obtained from the Protein Data Bank (www.rcsb.org): 52 PDB ID 1D4A,53 and PDB ID 3ZCF, respectively.54

We note that the smaller sensitivity observed in the liposome studies (30-fold enhancement vs 70 fold and >1,000 fold enhancements for SEC and chemical reduction studies, respectively) may be the result of partial probe reduction under the biological conditions explored. The higher polarity of the water-lipid interface where the probe is expected to sit, akin to that in acetonitrile,56,57 may also result in a smaller emission quantum yield for the probe, see Table 1 and discussion of SEC vs chemical reduction experiments, vide supra. Additionally, we note that O2 leakage may lead to a steady state condition that results in a lower intensity at the plateau as a result of ongoing regeneration of B-VKQ from B-VKQH2 . Consistent with the latter, we recorded a decrease of the fluorescence intensity over time in the enzymatic reduction assay after a plateau was initially reached. Upon degassing with additional argon, we observed a restoration of fluorescence intensity, before a new plateau was reached (Figure 6). This confirms oxygen diffuses back in the system causing auto-oxidation of the probe, further highlighting its extreme sensitivity to the redox environment.

Fluorescence (counts/s)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 12

Figure 6. Increase in fluorescence upon enzymatic reduction of B-VKQ by DT-diaphorase. Experiments were conducted after degassing with Ar, where the system was re-purged with argon to remove oxygen at the time indicated by the arrow.

CONCLUSION We have shown herein the synthesis and characterization of a two segment receptor-reporter fluorogenic vitamin K analogue B-VKQ, a desirable probe to monitor electron transfer and transport in model systems and biological structures. B-VKQ displays reversible turn-on fluorescence upon two-electron and two proton reduction of the menadione moiety to its menadiol counterpart. It retains the redox specificity and reversibility imparted from the menadione moiety as validated via a suite of chemical reactions and spectroelectrochemical studies. As such, it may find use in the study of nanomaterials relying on electron transport through films and interfaces applying single molecule spectroelectrochemistry (SM-SEC) techniques.47 Our own SEC studies provide preliminary results in this direction. Furthermore, B-VKQ shows high sensitivity, surpassing 1,000 fold fluorescence enhancement in non-polar systems, making it a suitable probe to conduct enzymology studies not only in bulk but also at the single molecule level, where signal to background ratios place a stringent constraint on the fluorescent contrast between the off and on forms of a fluorogenic probe. Important for bioimaging considerations, the biological compatibility of our probe was demonstrated upon reactions with DT-diaphorase (a naturally occurring quinone oxidoreductase) and via lipid membrane insertion. Given that the receptor segment preserves intact the electronic moiety of vitamin K3, we further postulate that B-VKQ may enable a better understanding of the role that vitamin K plays in key biological redox processes including photosynthesis and cellular respiration, and help elucidate the mechanism and pathological significance of redox reactions in biological systems.

3.0x104 additional Ar purging

2.0x10

4

1.0x104 0.0

0

100 200 300 Time (min)

400

8


Page 9 of 12

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of the American Chemical Society mosphere, with a minimum scan rate of 0.2 V s-1. The formal redox potential was calculated from the midpoint of the cathodic and anodic peak potentials observed in the cyclic voltammograms.

EXPERIMENTAL SECTION Materials. 8-Acetoxymethyl-2,6-diethyl-1,3,5,7-tetramethylpyrromethene fluoroborate (PM605) was purchased from Exciton, Inc. (Dayton, OH). HPLC-grade solvents for spectroscopy and column chromatography were purchased through Fischer Scientific. All chemical reagents were purchased from Sigma-Aldrich and were used without further purification. Water was purified by Millipore Milli-Q system.

Fluorescence Spectroelectrochemistry. The ITO coverslips (Evaporated Coatings Inc., 50 Ω/□; cleaned by 10 minutes sequential sonication in acetone, trichloroethylene and methanol) were platinized to improve the catalytic activity of the electrode. Platinum was deposited by spin coating 2 drops of ~ 20 µL of platinum precursor solution (Plastisol T, Solaronix) on coverslips while rotating at 500 rpm. The rotation was accelerated to 2000 rpm and held at that speed for 60 seconds to remove excess solution. The coverslips were then sintered at 450 °C for 30 minutes to form the transparent platinum coating. After cooling to allow for handling, a glass tube was glued with epoxy (Hardman®, McMaster-Carr) on top of the coverslip to form the sample chamber. The epoxy glue was cured by placing the samples in a 110 °C oven for ~ 30 minutes. The sample chamber was filled with 2 mL of the desired solution (500 nM analyte, 0.1 M Bu4NClO4 in dry acetonitrile) and sealed by a septum pierced with the reference (AgClcoated Ag wire) and counter (Pt wire) electrodes. Argon was bubbled through the solution for 1 hour in order to deaerate the solution and an argon-filled balloon was fixed on the sample during acquisition. The electrochemical cell was then mounted on the microscope (IX-71, Olympus). The electrochemical potential was controlled by a CHI600D electrochemical analyzer. The AgCl-coated Ag wire quasireference electrode used was calibrated with the ferrocene/ferrocenium redox couple using E0Fc+/Fc = + 0.64 V vs. NHE. All potentials reported are referenced to NHE.

Instrumentation. Absorption spectra were recorded using a Hitachi U-2800 UV-vis-NIR spectrophotometer. Room temperature luminescence spectra were recorded using a PTI QuantaMaster spectrofluorimeter using 1 cm × 1 cm quartz cuvettes and corrected for detector sensitivity. 3 mm × 3 mm quartz cuvettes were used for the fluorimeter enzymatic assays. A Biotek Synergy 2 multimode microplate reader was used to record the absorbance-time trajectories for the enzyme assays. 1H NMR and 13C NMR spectra were recorded on a Bruker AV500 instrument at 500 and 125 MHz, respectively. Electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) mass spectra were measured on a Bruker maXis impact. Voltammetric experiments were conducted with a computer-controlled CHI760C potentiostat. Computational Methods. All quantum mechanical calculations were performed using the Gaussian 09 package.31 HOMO and LUMO orbital energies were determined from the molecular geometries optimized at the B3LYP 631G(d) level.58 Orbitals were visualized using the Gaussview 5 package. Fluorescence Lifetime Studies. The fluorescence lifetime measurements were carried out using a Picoquant Fluotime 200 Time Correlated Single Photon Counting (TCSPC) setup employing an LDH 470 diode laser from Picoquant as the excitation source. The laser output was at 466 nm. The excitation rate was 10 MHz, and the laser power was adjusted to ensure that the detection frequency was less than 100 kHz. The laser was controlled by a PDL 88 B picosecond laser driver from Picoquant. Photons were collected at magic angle polarization.

The laser excitation was provided by a supercontinuum laser (WhiteLase SC-400-4, Fianium, Beverly, MA). A center excitation wavelength of 515 nm was spectrally separated from the broadband emission by a computer-controlled acousto-optical tunable filter (AOTF, Fianium) and further cleaned by ET650spx and HQ517/30x excitation filters (Chroma, Rockingham, VT). A Z514-633rdc dichroic mirror (Chroma) projected the pulsed laser excitation (20 MHz repetition rate, 0.13 µW out of the objective) towards the water-immersion objective (UPLSAPO 60XW, N.A. = 1.20, Olympus). The confocal observation volume was focused 20 µm above the electrode surface, as controlled by a piezoelectric objective scanner (PIFOC P-721 controlled by an E710, Physik Instrumente (PI), Auburn, MA). The laser excitation was blocked by a 514 nm Raman notch filter and the fluorescence emission was collected by the same objective, passed through a 50 µm diameter pinhole positioned at the image plane, and split onto two avalanche photodiode detectors (APDs; SPCM‐AQR‐14, Perkin Elmer Optoelectronics, Vaudreuil, QC) with a 50:50 non-polarizing beamsplitter in pseudo-cross-correlation format. The data acquisition, signal from the detectors and the piezo scanner were controlled and analyzed by software from PicoQuant (SymPhoTime).

Electrochemical Measurements. The electrochemical measurement were performed using a three-electrode system. The working electrode was a glassy carbon (GC) electrode (except for B-VKQMe2 whose CV was obtained under identical conditions but utilizing a Pt working electrode), a Pt wire was used as the counter electrode, and an Ag/AgNO3 non-aqueous electrode was used as the reference. A 0.1 M solution of tetrabutylammonium hexafluorophosphate in dry or wet (10.8M) acetonitrile was used as the electrolyte solvent in which the dye was dissolved to 1 mM. Concentrations were determined from the absorption and calculated extinction coefficients. The solutions also contained ferrocene with a concentration of 1 mM as an internal standard. The solutions were equilibrated with argon, and all measurements were conducted under inert at9



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Liposome Preparation. Aqueous solutions 20 mM in lipids were prepared as follows: 15.8 mg of DOPC was weighed in a dry vial and dissolved with a minimal amount of chloroform. The solvent was evaporated with a stream of argon while rotating the sample vial to create a thin film on the vial wall. The film was left under vacuum to remove excess solvent. After 1 h the aliquot of DOPC was hydrated with 1 mL of a pH 7.4, 50 mM Tris/HCl, yielding 20 mM lipid suspensions. The lipid suspension was subjected to three freeze-thaw-sonication-vortex cycles, where each cycle involved storing the vials with the solutions in dry ice for 4 min and then thawing at 37°C for 4 min, followed by 4 min sonication. After the third cycle, the lipid suspensions were extruded 16 times using an Avanti mini-extruder with one 100 nm polycarbonate membrane. Liposomes roughly 100 nm in diameter and each containing ca. 100 000 DOPC lipids were thus obtained.

Page 10 of 12

since the reduced probe rapidly oxidizes back to the nonemissive B-VKQ in air. The fluorescence assays were conducted in a 3 mm × 3 mm quartz cuvette. 50 mM Tris/HCl buffer, 1 mg/mL (~10 units/mL) of DT-diaphorase, and 200 µM NADPH were purged with argon and added to the cuvette. The initial fluorescence intensity was recorded. An aliquot of 200 μM of B-VKQ embedded into liposomes (20 mM in lipids) was also purged with argon. 7.5 µL was added to the cuvette in order to have a final concentration of 1 mM lipids and 10 µM probe. The intensity-time trajectory was then recorded for a period of 5.5 h at 1 s intervals at 37°C. Controls were also performed without the addition of probe (only liposomes) or DT-diaphorase, or NADPH, under the same conditions.

Supporting Information. Synthesis, 1H NMR, 13C NMR, and HRMS characterization, absorption and emission spectra of compounds, enzyme assay calculations and control experiments. This material is available free of charge via the Internet at http://pubs.acs.org.

In order to prepare liposomes containing the probe, the dye was extruded with the lipids to ensure proper embedding. 3.14 mg of DOPC in a dry vial and dissolved with minimum chloroform, then 13.3 μL of 3 mM stock of B-VKQ in chloroform was added to the vial. The solvent was evaporated as before to create a thin film on the vial wall and left under vacuum to remove excess solvent. The lipid-dye film was resuspended in 200 μL buffer to give 20 mM solution of lipids containing 200 μM of B-VKQ. The same freezethaw-sonication-vortex cycle and extrusion steps as above were performed.

[email protected]

The manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript.

Enzymatic Assays. a) Microplate Assays In order to test the enzymatic activity of DT-diaphorase, the NAD(P)H-dependent reduction of menadione was monitored in a coupled assay, under aerobic conditions. Reduced menadione, menadiol, in turn reduces cytochrome c, the absorbance of which can be followed at 550 nm (ε = 29500 M-1 cm-1)59. These experiments were conducted in a well-plate reader at 37°C. Measurements were made in 50 mM Tris/HCl pH 7.4, with 1 mg/mL (~10 units/mL) of DTdiaphorase, 200 µM NADPH (as the electron donor), 10 µM menadione (as the electron acceptor), and 30 µM cytochrome c. The reactions were initiated with addition of NADPH, and reached a plateau after less than one minute. The assays using the fluorogenic probe, B-VKQ, and monitoring cytochrome c absorbance, were conducted under the same conditions as before, where 10 µM of the probe was used. Menadione and B-VKQ were dissolved in dimethylsulfoxide for stock preparation due to their poor solubility in aqueous media. The final concentration of DMSO in the reaction well did not exceed 2%. Controls were also performed with the absence of enzyme, absence of probe, absence of NADPH, as well as testing all possible combinations of the assay components to ensure only the reaction between DT-diaphorase and B-VKQ in the presence of NADPH and cytochrome c gave rise to the increase in absorbance.

The authors declare no competing financial interest.

G.C. is grateful to the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Canadian Foundation for Innovation (CFI) for funding. M-.N.B is thankful to NSERC-CGS for a graduate scholarship; R.G. is thankful to NSERC for a postgraduate scholarship; and A.M.D. is thankful to the Drug Discovery and Training Program (CIHR) at McGill for a postdoctoral fellowship.

REFERENCES (1) Halliwell, B., Gutteridge, J. Free Radicals in Biology and Medicine; 4th ed. ed.; Oxford University Press, 2007. (2) Murphy, M. P. Biochem. J 2009, 417, 1. (3) Sazanov, L. A. Nat. Rev. Mol. Cell Biol. 2015, 16, 375. (4) Alberts, B.; Johnson, A.; Lewis, J.; Raff, M.; Roberts, K.; Walter, P. In Molecular Biology of the Cell; 4th edition ed.; Garland Science, Taylor&Francis Group: New York, 2002. (5) Kuhlbrandt, W. BMC Biol. 2015, 13, 89. (6) Swallow, A. J. Function of Quinones in Energy Conserving Systems; Academic Press: New York, San Francisco, London, 1982. (7) Garcia, A. A.; Reitsma, P. H. Vitamins and hormones 2008, 78, 23. (8) Kurosu, M.; Begari, E. Molecules 2010, 15, 1531.

b) Fluorimeter Assays. The enzymatic reduction of BVKQ was also monitored via the fluorescence of the reduced probe, B-VKQH2 . The system was purged with argon, 10


Page 11 of 12

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(9) Vos, M.; Esposito, G.; Edirisinghe, J. N.; Vilain, S.; Haddad, D. M.; Slabbaert, J. R.; Van Meensel, S.; Schaap, O.; De Strooper, B.; Meganathan, R.; Morais, V. A.; Verstreken, P. Science 2012, 336, 1306. (10) Arnon, D. I. Fed Proc 1961, 20, 1012. (11) Manzotti, P., de Nisi, P. Zocchi, G. Funct. Plant. Sci. Biotechnol. 2008, 2, 29. (12) Gong, X.; Gutala, R.; Jaiswal, A. K. Vitamins and hormones 2008, 78, 85. (13) Stafford, D. W. J Thromb Haemost 2005, 3, 1873. (14) Danziger, J. Clin J Am Soc Nephrol 2008, 3, 1504. (15) Grimm, J. B.; Heckman, L. M.; Lavis, L. D. Prog Mol Biol Transl Sci 2013, 113, 1. (16) Nadler, A.; Schultz, C. Angew. Chem. Int. Ed. Engl. 2013, 52, 2408. (17) Chan, J.; Dodani, S. C.; Chang, C. J. Nat Chem 2012, 4, 973. (18) Miller, E. W.; Bian, S. X.; Chang, C. J. J. Am. Chem. Soc. 2007, 129, 3458. (19) Kaur, A.; Kolanowski, J. L.; New, E. J. Angew. Chem. Int. Ed. Engl. 2016, 55, 1602. (20) Krumova, K.; Cosa, G. In Photochemistry; Albini, A., Fasani, E., Eds.; Royal Society of Chemistry: Cambridge, 2013; Vol. 41, p 279. (21) Oleynik, P.; Ishihara, Y.; Cosa, G. J. Am. Chem. Soc. 2007, 129, 1842. (22) Khatchadourian, A.; Krumova, K.; Boridy, S.; Ngo, A. T.; Maysinger, D.; Cosa, G. Biochemistry 2009, 48, 5658. (23) Krumova, K.; Friedland, S.; Cosa, G. J. Am. Chem. Soc. 2012, 134, 10102. (24) Krumova, K.; Greene, L. E.; Cosa, G. J. Am. Chem. Soc. 2013, 135, 17135. (25) Greene, L. E.; Godin, R.; Cosa, G. J. Am. Chem. Soc. 2016, 138, 11327. (26) de Silva, A. P.; Gunaratne, H. Q.; Gunnlaugsson, T.; Huxley, A. J.; McCoy, C. P.; Rademacher, J. T.; Rice, T. E. Chem. Rev. 1997, 97, 1515. (27) Loudet, A.; Burgess, K. Chem. Rev. 2007, 107, 4891. (28) Wood, T. E.; Thompson, A. Chem. Rev. 2007, 107, 1831. (29) Liu, X.-Y.; Long, Y.-T.; Tian, H. RSC Adv. 2015, 5, 57263. (30) Lincoln, R.; Greene, L. E.; Krumova, K.; Ding, Z.; Cosa, G. J. Phys. Chem. A 2014, 118, 10622. (31) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery Jr., J. A.; Peralta, J. E.; Ogliaro, F.; Bearpark, M. J.; Heyd, J.; Brothers, E. N.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A. P.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, N. J.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, Ö.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J.; Gaussian, Inc.: Wallingford, CT, USA, 2009. (32) Godin, R.; Liu, H. W.; Smith, L.; Cosa, G. Langmuir 2014, 30, 11138.

(33) Neuteboom, E. E.; Meskers, S. C. J.; Beckers, E. H. A.; Chopin, S.; Janssen, R. A. J. J. Phys. Chem. A 2006, 110, 12363. (34) Weaver, M. J. Chem. Rev. 1992, 92, 463. (35) Prince, R. C.; Dutton, P. L.; Bruce, J. M. FEBS Lett. 1983, 160, 273. (36) Krumova, K.; Cosa, G. J. Am. Chem. Soc. 2010, 132, 17560. (37) Hui, Y.; Chng, E. L.; Chng, C. Y.; Poh, H. L.; Webster, R. D. J. Am. Chem. Soc. 2009, 131, 1523. (38) Aguilar-Martínez, M.; Macías-Ruvalcaba, N. A.; Bautista-Martínez, J. A.; Gómez, M.; González, F. J.; González, I. Curr. Org. Chem. 2004, 8, 1721. (39) Gupta, N.; Linschitz, H. J. Am. Chem. Soc. 1997, 119, 6384. (40) Cannes, C.; Kanoufi, F.; Bard, A. J. Langmuir 2002, 18, 8134. (41) Mack, D. O.; Wolfensberger, M.; Girardot, J. M.; Miller, J. A.; Johnson, B. C. J. Biol. Chem. 1979, 254, 2656. (42) Bovicelli, P.; Borioni, G.; Fabbrini, D.; Barontini, M. Synth. Commun. 2008, 38, 391. (43) Heineman, W. R. J. Chem. Educ. 1983, 60, 305. (44) Palacios, R. E.; Chang, W. S.; Grey, J. K.; Chang, Y. L.; Miller, W. L.; Lu, C. Y.; Henkelman, G.; Zepeda, D.; Ferraris, J.; Barbara, P. F. J. Phys. Chem. B 2009, 113, 14619. (45) Yilmaz, I. Transition Met. Chem. 2007, 33, 259. (46) Pinyayev, T. S.; Seliskar, C. J.; Heineman, W. R. Anal. Chem. 2010, 82, 9743. (47) Godin, R.; Cosa, G. J. Phys. Chem. C 2016. (48) The enhancement is calculated after the fluorescence intensity-time trajectory of B-VKQ in the presence of acid under applied bias has been subtracted by that of the blank electrolyte solution. (49) Iyanagi, T.; Yamazaki, I. Biochim. Biophys. Acta 1970, 216, 282. (50) Cui, K.; Lu, A. Y.; Yang, C. S. Proc Natl Acad Sci U S A 1995, 92, 1043. (51) Tedeschi, G.; Chen, S.; Massey, V. J. Biol. Chem. 1995, 270, 1198. (52) Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T. N.; Weissig, H.; Shindyalov, I. N.; Bourne, P. E. Nucleic Acids Res. 2000, 28, 235. (53) Faig, M.; Bianchet, M. A.; Talalay, P.; Chen, S.; Winski, S.; Ross, D.; Amzel, L. M. Proc Natl Acad Sci U S A 2000, 97, 3177. (54) Rajagopal, B. S.; Edzuma, A. N.; Hough, M. A.; Blundell, K. L.; Kagan, V. E.; Kapralov, A. A.; Fraser, L. A.; Butt, J. N.; Silkstone, G. G.; Wilson, M. T.; Svistunenko, D. A.; Worrall, J. A. Biochem. J 2013, 456, 441. (55) Qian, Y.; Karpus, J.; Kabil, O.; Zhang, S. Y.; Zhu, H. L.; Banerjee, R.; Zhao, J.; He, C. Nat Commun 2011, 2, 495. (56) Träuble, H.; Overath, P. Biochim. Biophys. Acta 1973, 307, 491. (57) Schlamadinger, D. E.; Gable, J. E.; Kim, J. E. J. Phys. Chem. B 2009, 113, 14769. (58) Mendez-Hernandez, D. D.; Tarakeshwar, P.; Gust, D.; Moore, T. A.; Moore, A. L.; Mujica, V. J. Mol. Model. 2013, 19, 2845. (59) van, G. B.; Slater, E. C. Biochim. Biophys. Acta 1962, 58, 593.

11



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

.

12


Page 12 of 12

Monitoring Chemical and Biological Electron Transfer Reactions with

Recommend Documents